Platelet aggregation inhibitory agents and intermediate therefor

ABSTRACT

This invention relates to certain N,N&#39;-bis(nipecotyl)piperazines and their use as platelet aggregation inhibtory agents.

The present application is a divisional application of U.S. Ser. No.909,135 filed on Sept. 18, 1986 now U.S. Pat. No. 4,798,830. U.S. Ser.No. 909,135 is a divisional application of Ser. No. 571,326 filed Jan.6, 1987, now U.S. Pat. No. 4,634,209 which is a divisional applicationof Ser. No. 368,863, filed on Apr. 15, 1982, now U.S. Pat. No.4,443,450.

RELATED U.S. APPLICATION DATA

Application Ser. No. 347,037, titled PLATELET AGGREGATION INHIBITORYAGENTS and filed on Feb. 8, 1982 in the names of Andrew Lasslo, RonaldP. Quintana and Marion Dugdale, contains subject matter which is relatedto the invention described and claimed herein. The disclosure of thatapplication is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to certain novel compounds useful asplatelet aggregation inhibitory agents and other novel compounds usefulas intermediates in the preparation thereof.

Thromboembolic disorders have been shown to be directly related to thesusceptibility of blood platelets to adenosine diphosphate (ADP) andthrombin induced platelet aggregation and to the otheradhesion-release-aggregation chain reactions. Animals wearing prostheticdevices or whose blood is exposed to biomaterials during renal dialysis,blood oxygenation, cardiac catheterization, etc., are especiallypredisposed to thromboembolic disorders.

The susceptibility of animal blood platelets to aggregation has alsobeen shown to be directly related to the platelet membrane stability.

Certain chemical compounds are known to inhibit platelet aggregation.Thus, aspirin, sulfinpyrazone and dipyridamole are known plateletaggregation inhibiting agents. See Quintana et al, Thromb. Res., Vol.20, pages 405-415 (1980); Cucuianu et al, J. Lab. Clin. Med., Vol. 77,pages 958-974 (1971) and Zucker et al. J. Lab. Clin Med., Vol. 76, pages66-75 (1970).

It is an object of the present invention to provide a composition andmethod for inhibiting blood platelet aggregation thereby being usefulfor the treatment of thromboembolic disorders.

It is further an object of the present invention to provide a novelclass of compounds having platelet aggregation inhibiting activity andplatelet membrane stabilization characteristics useful for the treatmentof thromboembolic disorders.

SUMMARY OF THE INVENTION

The present invention is predicated on the discovery that compounds ofthe following structural formulae exhibit blood platelet aggregationinhibiting activity when administered to animals in need thereof:

(A) ##STR1## Wherein R is ##STR2## and R₁ is alkyl;

(B) ##STR3## (C) ##STR4## Wherein R₂ is ##STR5## and R₃ is alkyl or H;

(D) R₄ --H₂ C--(CH₂)_(n) --CH₂ --R₅ Wherein

R₄ is ##STR6## R₅ is H or R₄, n=4 or 8 when R₅ is H, and

n=8 when R₅ is R₄ ;

(E) R₆ --H₂ C--(CH₂)_(n) --CH₂ --R₇ Wherein

R₆ is ##STR7## R₇ is R₆ or H, R₈ is H or alkyl,

R₉ is H or lower alkyl, and

n is 0, 2, 4, 6 or 8; and

(F) Addition salts thereof with pharmaceutically acceptable acids.

The invention also relates to methods for the inhibition of bloodplatelet aggregation comprising the administration to an animal in needthereof a blood platelet aggregation inhibitory amount of a compound ofthe above formulae.

The invention also relates to a pharmaceutical composition in unitdosage form suitable for usage in the above described method comprisinga pharmaceutically acceptable carrier and a blood platelet aggregationinhibitory amount of a compound of the above structural formulae.

The compounds are preferably compounded in unit dosage form withpharmaceutically acceptable carriers such as, e.g., (1) tablets:lactose, starch 5%--acacia 2% in water, corn starch, calcium stearate;(ii) capsules: lactose; (iii) parenterals: sterile solid or constitutedaqueous solution, including antibacterial, antioxidant, chelating andbuffering agents; (iv) suppositories: cocoa butter, and administeredorally, parenterally, or rectally to animals in need thereof.

The invention also relates to certain novel compounds having little orno platelet aggregation inhibiting activity but which are useful asintermediates in the preparation of the above-described activecompounds.

The intermediate compounds have the structural formulae:

(A) R₁₀ --CH₂ --(CH₂)_(n) --CH₂ --R₁₁ Wherein

R₁₀ is ##STR8## R₁₁ is H, R₁₂ is alkyl, and

n is 6;

(B) R₁₃ --CH₂ --(CH₂)_(n) --CH₂ --R₁₄ Wherein

R₁₃ is ##STR9## R₁₄ is H or R₁₃, R₁₅ is alkyl or H,

R₁₆ is alkyl, and

n is 4 or 8;

(C) ##STR10## Wherein n is 4 or 8; and ##STR11## Wherein n is 8;

(D) ##STR12## Wherein R₁₇ is ##STR13## R₁₈ is alkyl, and R₁₉ is alkyl;

(E) ##STR14## Wherein R₂₀ is ##STR15## and R₂₁ is alkyl, and

(F) Addition salts thereof with pharmaceutically acceptable acids.

With respect to the term, "alkyl", the preferred groups, in the amidefunction, are the lower alkyl groups, i.e. alkyl groups having one tosix carbon atoms.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts inhibition of ADP-induced human blood plateletaggregation effected by Example 37 in comparison with aspirin.

FIG. 2 depicts inhibition of thrombin-induced human blood plateletaggregation effected by Example 37 in comparison with aspirin andchlorpromazine.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the identification of aparticular set of structural features in certain chemical compoundswhich appear ideal for the constructive penetration of the human bloodplatelet plasma membrane's lipid bilayer and for effecting membranestabilization. Quintana et al [Thromb. Res., Vol. 22, pages 665-680(1981)] conceived of and synthesized certain carbamoylpiperidinoalkanesand -aralkanes incorporating these features, remarkably suited for thepenetration to, and subsequent interaction with, negatively chargedphospholipids which are prominently instrumental in the plateletaggregation process. Moreover, independently, the cationic form of thepenetrated compounds can be envisioned as blocking the mobiliation ofCa++ ions by stimuli up to given levels of intensity thereby increasingthe threshold effecting Ca++-dependent phospholipase A₂ and/orphospholipase C activity. It was further substantiated that tertiaryamide and tertiary amino groups elicit optimal activity. The tertiaryamines in the piperidine moieties have a particularly pivotal function(Quintana, Lasslo and Dugdale, Biophys. J., Vol. 37, pages 130-133(1982)). Being generally subject to broad variances in protonation theycan assume in carefully tailored molecules appropriate hydrophobiccharacter for the penetration of the platelet membrane's lipid bilayerwithout interfering with the subsequent generation of adequate cationicspecies (Quintana, Lasslo, Dugdale and Goodin, Thromb. Res., Vol. 22,pages 665-680 (1981); Quintana Lasslo and Dugdale, Biophys. J., Vol. 37,pages 130-133 (1982)). In addition, the compounds incorporate othermolecular components to enhance their lipophilic character, asnecessary, for tuning hydrophobic bonding and, thereby, facilitatingtheir action (Quintana, Lasslo and Queen, Chem.-Biol. Interactions, Vol.38, 135-144 (1982)).

In studies of individual phospholipids (Quintana, Lasslo and Greer,Thromb. Res., Vol. 24, pages 379-395 (1981)), involving the matchingfeatures and serving as prototypes of actual platelet membraneconstituents, the compounds disclosed in the above-identified U.S.Patent Application Ser. No. 347,033 registered strong interaction withphosphatidylserine (PS) and phosphatidylinositol (PI), none withphosphatidylcholine (PC), and barely interacted withphosphatidylethanolamine (PE). These results are significant sincephosphatidylserine and phosphatidylinositol are deemed to constitute thedominant components of platelet factor 3, the catalytic surface forelements of the platelet aggregation process (Caen et al, Platelets:Physiology and Pathology: New York, Stratton International, 1977; pages17-18) (cf. Bode et al, Ann. N.Y. Acad. Sci., Vol. 370, pages 348-358(1981)). Lewis and Majerus (J. Clin. Invest., Vol. 48, pages 2114-2123(1969)) and Zwall (Biochim. Biophys. Acta., Vol. 515, pages 163-205(1978)) have stressed the major role of PS in aggregation, and Gerrardet al (Calcium Mobilization; in Platelets in Biology and Pathology--2,J. L. Gordon, ed., New York, Elsevier/North Holland, 1981; page 430)depicts PI in a "loaded gun" arrangement, triggered among the "earliestevents" in ADP and thrombin stimulated platelets.

The novel active compounds described herein and methods for theirpreparation and use are illustrated by the following non-limitingexamples:

Representation Procedure A

N,N'-Bis(nicotinoyl)-piperazine (Example 34) was prepared by a procedureadapted from the one reported by Badgett et al. (J. Am. Chem. Soc., Vol.67, pages 1135-1138 (1945). Thus, thionyl chloride (71.4 g, 0.600 mole)was added dropwise to a cold stirred mixture of 73.9 g (0.600 mole) ofnicotinic acid, 94.9 g (1.20 moles) of pyridine and 196 ml of toluene.After the reaction mixture was gradually heated to 90° C. and maintainedat this temperature for 1-2 hr, 25.8 g (0.300 mole) of piperazinedissolved in 120 ml of hot toluene was dispensed gradually into thereaction mixture from a dropping funnel in which the solution wasmaintained hot by circulating steam or hot water through the funnel'sjacket. The stirred mixture was maintained at 90° C. for an additional1-2 hr, after which it was poured into 500 ml of ice water and the pHadjusted to 9.0 with sodium carbonate. The resulting suspension wasextracted with a total of 950 ml of chloroform, the chloroform extractdried over anhydrous magnesium sulfate, the drying agent was removed,the filtered solution decolorized by boiling with charcoal and thecharcoal removed by filtration through Celite. The filtrate was reducedin volume to 400 ml whereupon crystallization ensued, yielding theproduct with properties indicated in Table 1. As indicated in thetabulation, Ross (J. Med. Chem., Vol. 10, pages 257-259 (1967)) reportedthe preparation of this compound by a different procedure withoutrecording the yield. The present method produced 42.2% of analyticalgrade product.

Representative Procedure A₁

Same as Procedure A, except that the amine was added directly to thenicotinic acid - thionyl chloride - pyridine mixture in the absence oftoluene.

Representative Procedure B

1,10-Bis[N-1-methylnicotinoyl)amino]decane diiodide (Example 27) wasprepared employing an adaptation of the procedure described by Lassloand Kimura (J. Am. Pharm. Assn., Sci. Ed., vol. 39, pages 43-46 (1950))(cf. Karrer, Ber., Vol. 49, pages 2057-2079 (1916); Keller and Bernhard,Arch. Pharm., vol. 263, pp. 401-424 (1925); Pyman, J. Chem. Soc., vol.113, pages 222-234 (1918). 1,10-Bis(N-nicotinoylamino)decane (13.0 g,0.034 mole) was dissolved in 459 ml of 95% ethanol and 292 ml of water,96.5 g (0.680 mole) of methyl iodide was added, and the mixture wasstirred for 5 min. Sodium carbonate (31.5 g, 0.297 mole) was thenintroduced, and the contents of the reaction vessel were heated at42°-45° C. for 8 hr. The reaction mixture was concentrated, in vacuo, byremoving excess methyl iodide, ethanol and most of the water. Theresulting aqueous slurry was extracted with 250 ml of 95% ethanol. Thesolvent was removed under vacuum. The crude product (24.4 g,quantitative crude yield) was recrystallized from 95% ethanol, yieldingthe compound with the physical properties described in Table 1.

Representative Procedure B₁

Same as Procedure B except that the compound was extracted from theaqueous slurry with chloroform.

Representative Procedure B₂

Same as Procedure B except that the compound was extracted from theaqueous slurry with boiling absolute ethanol, charcoal treated, filteredthrough Celite and allowed to crystallize from the same.

Representative Procedure B₃

Same as Procedure B except that it is conducted without sodiumcarbonate, and anhydrous benzene was substituted for the aqueous ethanolreaction vehicle (cf. Lasslo. Marine and Waller, J. Org. Chem., Vol. 21,pages 958-960 (1956); Lasslo and Waller, J. Org. Chem., Vol. 22, pages837-838 (1957).

Representative Procedure B₄

Same as Procedure B except that it is conducted without sodiumcarbonate, and absolute ethanol was substituted for the aqueous ethanolreaction vehicle.

Representative Procedure B₅

Same as Procedure B except that it is conducted without sodiumcarbonate, and ethanol-acetone was substituted for the aqueous ethanolreaction vehicle (cf. Quintana, Smith and Lorenzen, J. Pharm. Sci., Vol.54, pages 785-787 (1965)).

Representative Procedure B₆

Same as Procedure B except that it is conducted without sodiumcarbonate, and quaternization was effected by heating the amine andalkyl halide reactants, without any solvent, under a nitrogen atmosphere(cf. Few, Gilby, Ottewill and Parreira, J. Chem. Soc., pages 4712-4713(1958)).

Representative Procedure C

1,10-Bis[N-1-methylnipecotoyl)amino]decane dihydriodide (Example 28) wasprepared in accordance with the procedure described by Lasslo et al forrelated compounds (Lasslo, Marine and Waller, J. Org. Chem., Vol. 21,pages 958-960 (1956); Lasslo and Waller, J. Org. Chem, Vol. 22, pages837-838 (1957)). Thus, 12.0 g (0.018 mole) of1,10-bis[N-(1-methylnicotinoyl)amino]decane diiodide was dissolved in amixture of 125 ml of absolute ethanol and 125 ml of water, and subjectedto hydrogenation (at maximum pressures of 45-50 psi and at ambienttemperature) in the presence of 1.0 g of platinum oxide (Adams'catalyst). When hydrogen uptake ceased, the catalyst was removed byfiltration and the solvent by distillation under reduced pressure.Subsequent to drying at 75° C./0.020 mm Hg for 2 hr, the residueconstituted the analytical sample (11.8 g, 96.7%) with the propertiesdescribed in Table 1. In other instances, the products so obtained werepurified by recrystallization from appropriate solvents.

Representative Procedure C₁

Same as Procedure C except that a specific ratio of ethanol to water wasrequired for solubilization of the unsaturated intermediate. E.g., 4.0 g(0.007 mole) of α,α'-bis(6-hydroxyquinolinium)-p-xylene dibromide(Example 4) was dissolved in 150 ml of boiling water contained in theParr hydrogenation vessel. Immediately after cessation of heating, 95%ethanol was added to the hot solution increasing the volume to a totalof 275 ml. Platinum oxide (0.5 g) was added and the contents of thereaction vessel were maintained at 50° C., employing a Parr heatingmantle and thermistor probe, during the entire period of hydrogenation.

Representative Procedure D

1,2-Bis(N-nipecotoyl-N-methylamino)ethane dihydrobromide (Example 39).In accordance with a procedure described by Lasslo et al (Lasslo, Marineand Waller, J. Org. Chem., Vol. 21, pages 958-960 (1956); Lasslo andWaller, J. Org. Chem., Vol. 22, pages 837-838 (1957)); 10.0 g (0.036mole) of 1,2-bis(N-nicotinoyl-N-methylamino)ethane (Example 38) wasdissolved in 50 ml of water. To the solution was added 12.0 ml of 48%HBr. The solution was filtered through Celite and, upon dilution to atotal volume of 275 ml, subjected to hydrogenation (at maximum pressuresof 45-50 psi and at ambient temperature) in the presence of 1.0 g ofplatinum oxide (Adams' catalyst). When hydrogen uptake ceased, thecatalyst was removed by filtration and the solvent by distillation underreduced pressure. The residue (16.7 g, quantitative yield) was purifiedby dissolving in boiling absolute ethanol with activated charcoal. Thecharcoal was removed by filtration through Celite and the whitecrystalline product obtained, after removal of the solvent bydistillation under reduced pressure, was highly hygroscopic.

The physical characteristics and analyses of the examples preparedaccording to the above procedures (referenced in the Method column) areset forth in Table 1. Some inactive compounds (and their intermediates)are included for comparison with the active entities since therelationships between molecular constitution and platelet aggregationinhibitory potency are important.

    TABLE 1      ANALYSES METHOD of C, % H, % N, % Br, % I, % EXAMPLE COMPOUND PREPARATIO     N M.P. °C. or (B.P. °C.) or [n.sub.D.sup.25 ] Calcd. Found C     alcd. Found Calcd. Found Calcd. Found Calcd. Found       13 N,N-Dibutylnicotin- A (129.0-134.0/ 71.76 71.87 9.46 9.40 11.95     12.02 -- -- -- --  amide (Intermediate.sup.1)  0.03 mm Hg) 23 1-Methyl-3-     (N,N-di- B.sub.1,C 98.5-99.0 47.12 47.09 8.17 8.16 7.33 7.40 -- -- 33.19     33.19  butylcarbamoyl)piper-  idine Hydriodide 20 N-Methyl-N-octylnico-     A.sub.1 (155.0-156.0/ 72.54 72.65 9.74 9.77 11.28 11.26 -- -- -- --     tinamide  0.10 mm Hg)  (Intermediate) 21 N-Methyl-N-octylnipe- C     (148.0-148.5/ 70.82 70.93 11.88 12.00 11.01 11.02 -- -- -- --  cotamide     0.15 mm Hg) 22 1-Methyl-3-(N-methyl- B.sub.1,C [1.5305] 48.49 48.35 8.39     8.28 7.07 7.03 -- -- 32.02 32.14  N-octylcarbamoyl)pi-  peridinc     Hydriodide 29 1,6-Bis(N-nicotinoyl- A.sub.1 165.0-166.5 66.24 66.23 6.79     6.89 17.17 17.08 -- -- -- --  amino)hexane  (Intermediate.sup.2) 26     1,10-Bis(N-nicotinoyl- A 168.5-168.9 69.08 68.82 7.90 7.80 14.65 14.45     -- -- -- --  amino)decane  (Intermediate.sup.3) 27 1,10-Bis[N-(1-methyl-     B 146.0-147.5 43.26 43.39 5.44 5.59 8.41 8.32 -- -- 38.09 38.17     nicotinoyl)amino]-  decane Diiodide 28 1,10-Bis[N-(1-methyl- C 98.5-99.5     42.49 42.30 7.13 7.20 8.26 8.05 -- -- 37.41 37.45  nipecotoyl)amino]-     decane Dihydriodide 33 1,10-Bis(N-methylpiper- B.sub.1 267.0-269.0 44.60     44.60 7.83 7.86 4.73 4.74 -- -- 42.84 42.93  idinium)decane Diiodide 32     α,α '-Bis(N-nicotinoyl- A.sub.1 173.0-174.0 69.35 69.21 5.24     5.23 16.17 16.15 -- -- -- --  amino)-m-xylene  (Intermediate) 19     α,α'-Bis[3-(N-decylcar- C.sub.1 203.0-203.5 59.99 59.98 9.06     9.03 7.00 6.98 19.95 19.74 -- --  bamoyl)piperidino]-p-  xylene Dihydrobr     omide 14 α,α'-Bis[3-(N,N-dibutyl- B.sub.5,C 256.5-257.5     58.06 58.16 8.66 8.69 7.52 7.51 21.46 21.58 -- --  carbamoyl)piperidino]-       p-xylene Dihydrobromide 34 N,N'-Bis(nicotinoyl)- A 201.5-202.5 64.85     64.75 5.44 5.57 18.91 18.91 -- -- -- --  piperazine  (Intermediate.sup.4)      37 N,N'-Bis(1-decylnipeco- C.sub.1 279.0-280.0 51.18 50.97 8.35 8.26     6.63 6.73 -- -- 30.04 30.16  toyl)piperazine  Dihydriodide 10 1-Hexyl-6-h     ydroxyquino- B.sub.6 92.5-93.5 50.43 50.19 5.64 5.58 3.92 3.87 -- --     35.52 35.42  linium Iodide  (Intermediate) 11 1-Hexyl-6-hydroxy-1,2,3, C     152.5-153.5 49.87 49.96 6.70 6.73 3.88 3.87 -- -- 35.13 35.18  4-tetrahyd     roquinoline  Hydriodide  8 1-Decyl-6-hydroxyquino- B.sub.6 87.0-89.5     55.21 55.23 6.83 6.99 3.39 3.37 -- -- 30.70 30.73  linium Iodide     (Intermediate)  9 1-Decyl-6-hydroxy-1,2, C 144.0-145.0 54.68 54.72 7.73     7.72 3.36 3.35 -- -- 30.41 30.61  3,4-tetrahydroquinoline  Hydriodide 12     1-Tetradecyl-6-hydroxy- B.sub.6,C 148.0-149.0 58.35 58.30 8.51 8.67 2.96     2.93 -- -- 26.80 26.84  1,2,3,4-tetrahydro-  quinoline Hydriodide  7     1,10-Bis(6-hydroxy-1, C.sub.1 217.0-218.0 48.57 48.56 6.11 6.24 4.05     3.98 -- -- 36.65 36.59  2,3,4-tetrahydroquino-  lino)decane Dihydriodide      4 α,α'-Bis(6-hydroxyquino- B.sub.5 310.0-317.0 56.34 56.15     4.00 4.13 5.05 4.99 28.83 28.87 -- --  linium)-p-xylene Dibro-  (decompos     ition)  mide  (Intermediate)  5 α,α'-Bis(6-hydroxy-1,2,     C.sub.1 287.0-288.5 55.53 55.42 5.38 5.51 4.98 4.85 28.42 28.27 -- --     3,4-tetrahydroquinolino)-  p-xylene-Dihydrobromide      2 α,α'-Bis(1,2,3,4-tet- C.sub.1 298.0-299.0 58.88 58.76     5.70 5.78 5.28 5.27 30.13 29.99 -- --  rahydroisoquinolino)-  p-xylene     Dihydrobro-  mide.sup.5 36 N,N-Bis(1-decylnico- B.sub.4 250.0-251.5     51.93 51.82 7.02 7.02 6.73 6.67 -- -- 30.48 30.41  tinoyl)piperazine     Diiodide  (Intermediate) 18 α,α'-Bis[3-(N-decyl- B.sub.5     255.5-256.2 60.91 60.92 7.67 7.56 7.10 7.03 20.26 20.34 -- --  carbamoyl)     pyridinium]-  p-xylene Dibromide  (Intermediate)      1 α,α'-Bis(isoquinolin- B.sub.5 350.0-352.0 59.79 59.83     4.25 4.28 5.36 5.29 30.60 30.45 -- --  ium)-p-xylene Dibro-  (decompositi     on)  mide  (Intermediate) 16 α,α'-Bis[3-(N,N-didecyl- C     225.0-227.0 66.44 66.55 10.44 10.42 5.18 5.23 14.78 14.78 -- --     carbamoyl)piperidino] -  p-xylene Dihydrobromide 43 1,6-Bis[N-(1-decylnip     e- B.sub.4,C 61.0-62.0 55.97 55.95 9.39 9.26 5.68 5.77 -- -- 25.71 25.73      cotoyl)-N-butylamino]-  hexane Dihydriodide 42 1,6-Bis[N-(1-decylnipe-     B.sub.4,C 63.0-64.5 53.21 52.99 8.93 8.89 6.21 6.26 -- -- 28.11 27.95     cotoyl)-N-methylamino]-  hexane Dihydriodide      3 α,α'-Bis(quinolinium)- B.sub.5 360.0° 59.79 59.79     4.25 4.27 5.36 5.35 30.60 30.62 -- --  p-xylene Dibromide  (decomposition     )  (Intermediate.sup.6) 17 N-decylnicotinamide A 71.0-72.0 -- -- -- --     -- -- -- -- -- --  (Intermediate.sup.7)  6 1,10-Bis(6-hydroxy- B.sub.6     256.0-256.5 -- -- -- -- -- -- -- -- -- --  quinolinium)decane  Diiodide     (Intermediate.sup.8) 15 α,α'-Bis[3-(N,N-di- A.sub.1,B.sub.5     139.0-140.0 67.40 67.62 9.43 9.24 5.24 5.26 14.95 14.75 -- --  decylcarba     moyl)pyri-  dinium]-p-xylene Di-  bromide  (Intermediate) 30 1,6-Bis[N-(1     -methyl- B.sub.2 197.0-198.0 39.36 39.20 4.62 4.68 9.18 9.14 -- -- 41.60     41.60  nicotinoyl)amino]hex-  ane Diiodide  (Intermediate) 31 1,6-Bis[N-(     1-methyl- C 112.0-113.5 38.66 38.64 6.33 6.30 9.02 9.06 -- -- 40.85     40.73  nipecotoyl)amino]hex-  ane Dihydriodide 24 1-Methyl-3-(N-decyl-     B.sub.2 119.5-120.5 50.50 50.55 7.23 7.25 6.93 6.90 -- -- 31.39 31.37     carbamoyl)pyridinium  Iodide  (Intermediate) 25 1-Methyl-3-(N-decyl- C     55.0-56.0 49.76 49.70 8.60 8.52 6.83 6.79 -- -- 30.92 30.78  carbamoyl)pi     peridine  Hydriodide 35 N,N'-Bis(nipecotoyl)- D 319.0-319.5 40.87 40.83     6.43 6.41 11.91 11.84 33.98 33.87 -- --  piperazine Dihydro-  bromide 38     1,2-Bis(N-nicoti- A 143.8-144.8 64.41 64.39 6.08 6.05 18.78 18.66 -- --     -- --  noyl-N-methylamino)-  ethane  (Intermediate.sup.9) 39 1,2-Bis(N-ni     pecotoyl- D 157.0-158.5 40.69 40.66 6.83 6.94 11.86 11.89 33.84 33.83 --     --  N-methylamino)ethane  Dihydrobromide 40 1,2-Bis[N-(1-decylni-     B.sub.4,C 98.0-99.0 51.06 51.18 8.57 8.62 6.61 6.83 -- -- 29.97 29.91     pecotoyl)-N-methyla-  mino]ethane Dihy-  driodide 41 1,2-Bis[N-(1-hexyl-     B.sub.4,C 125.0-126.0 45.78 45.73 7.68 7.67 7.63 7.68 -- -- 34.55 34.59     nipecotoyl)-N-methyl-  amino]ethane Dihydri-  odide 44 1,6-Bis[N-(1-hexyl     - B.sub.4,C 72.0-73.0 52.17 52.15 8.76 8.76 6.40 6.35 -- -- 29.01 28.73     nipecotoyl)-N-butyl-  amino]hexane Dihydri-  odide     .sup.1 The preparation of this intermediate, using a different procedure,     was reported by Bald (Chem. Scr., Vol. 13, page 47 (1979)). No physical     properties were cited in the accessible literature.     .sup.2 This intermediate was apparently prepared, using a similar     procedure, by the Chugai Pharmaceutical Company (Jpn. Kokai Tokkyo Koho     80-81,861/1980; Chem. Abstr. Vol. 94, 139627u (1981). No physical     properties were cited in the accessible literature.     .sup.3 This intermediate was apparently prepared, using a similar     procedure, by the Chugai Pharmaceutical Company (Jpn. Kokai Tokkyo Koho     80-81,861/1980; Chem. Abstr., Vol. 94, 139627u (1981). No physical     properties were cited in the accessible literature.     .sup.4 This intermediate has been prepared by Ross (J. Med. Chem., Vol.     10, pages 257-259 (1967)), m.p. 201° C.     .sup.5 Mandelbaum and Bel (Adv. Mass. Spectrom., Vol. 8A, pages 828-831     (1980), reported the synthesis of this compound but did not cite a meltin     point or other physicochemical characteristics. The compound is inactive,     but its bis(6hydroxy-1,2,3,4-tetrahydroquinoline) analog (Example 5)     registered significant effect as an inhibitor of ADPinduced platelet     aggregation.     .sup.6 This compound is an intermediate for the tetrahydroderivative.     .sup.7 Literature, m.p. 72.1-72.4° C. (Badgett, Provost, Ogg and     Woodward, J. Am. Chem. Soc., Vol. 67, pages 1135-1138 (1945)).     .sup.8 It was authenticated through its tetrahydroderivative, compound of     Example 7.     .sup.9 This intermediate has been prepared by Newkome and Kawato (J. Org.     Chem., Vol. 45, pages 629-632 (1980)); m.p. 140.5-141.5° C.

The following samples illustrate the determination of the plateletaggregation inhibiting activity of the compounds of the invention.

Adenosine disphophate (ADP) used to induce platelet aggregation wasemployed as the sodium salt. A 10 mM stock solution was prepared freshbefore each use in modified Tyrode's buffer and working dilutions wereprepared with modified Tyrode's buffer immediately prior to use. Thebuffer contained NaCl (137.00 mM), KCl (2.70 mM), NaHCO₃ (11.90 mM),NaH₂ PO₄.H₂ O (0.36 mM) and glucose (5.60 mM) in redistilled water.Adjustment to pH 7.4 was effected by addition of 1N HCl. A solution ofhighly purified human α-thrombin (10,624 units/ml) was stored at -70° C.in 10 μl aliquots. The latter were diluted with modified Tyrode's bufferto a concentration of 10.0 units/ml immediately prior to use.

Venous blood for the examples set forth below was collected in plasticsyringes from healthy male volunteers (aged 21-30 years) who has fastedovernight and had abstained from all medications, alcohol, tobacco andcaffeine for a period of at least one week prior to donations. Inexperiments employing ADP as the aggregation-inducing agent, the bloodwas transferred into siliconized centrifuge tubes containing 3.2% sodiumcitrate (blood/citrate ratio 8:1); in thrombin-induced aggregations,3.8% sodium citrate (blood/citrate ratio 9:1) was used. The citratedwhole blood was centrifuged at 120×g for 15 minutes at 23° C., yieldingplatelet-rich plasma (PRP); platelet-poor plasma (PPP) was obtained bycentrifugation of citrated whole blood at 1,100×g for minutes at 23° C.The platelet count of PRP was determined and adjusted to a final countof 300,000 platelets per mm³ by dilution with autologous PPP.(Occasionally, blood from a given donor yielded PRP with a count lowerthan the stipulated figure; however, this was usually greater than285,000, and never less than 250,000 platelets per mm³.) The plasma soobtained was transferred in 1.2-ml aliquots to siliconized glass tubes,by means of a siliconized Pasteur pipet. In order to maintain plasma pHin the appropriate range, the air in the tubes was displaced gently (1minute) with a 5% CO₂ - 95% air (v/v) mixture and the tubes sealed withParafilm according to the method of Han et al, Br. J. Haematol. Vol. 26,pages 373-389 (1974). The plasma was maintained at 37° C. in water bathuntil used in the aggregation experiments.

Assays of platelet aggregation were performed at least in duplicate,using plasma acquired from different donors, employing a methoddeveloped by Quintana et al (Quintana et al, Thromb. Res., Vol. 22,pages 665-680 (1981); Quintana et al, Thromb. Res., Vol. 20, pages405-415 (1980) (cf. Born, Nature, Vol. 194, pages 927-929 (1962) andMustard et al, J. Lab. Clin. Med., Vol. 64, pages 548-559 (1964)).

Initially, in each experiment, 0.45-ml aliquots of PRP were placed insiliconized cuvettes and stirred (1,100 rpm) in the aggregometer at 37°C. to ascertain the absence of spontaneous aggregation. Appropriate ADPsolutions (50 μl) were subsequently injected using a Hamilton microlitersyringe to determine the minimal concentration eliciting maximalbiphasic aggregation. This ranged from 2 μM to 8 μM (cf. Quintana et al,Thromb. Res., Vol. 22, pages 665-680 (1981); Quintana et al, Thromb.Res., Vol. 20, pages 405-415 (1980)). In each case, the concentration ofADP so determined was used in eliciting aggregation throughout eachspecific set of aggregometric evaluations. In all thrombin-inducedaggregation experiments the same stimulus was applied; this constitutedinjection of 50 μl of the solution containing 10.0 units ofα-thrombin/ml (affording a final concentration of 1.0 unit ofα-thrombin/ml PRP).

0.5 μl of a solution of the evaluant compound in redistilled 95% ethanolwas injected into a stirred (1,100 rpm) 0.45-ml aliquot of plasma in asiliconized cuvette in the aggregometer-well (37° C.). After 15 seconds,the cuvette was transferred to an incubator (also at 37° C.) and thecontents held at this temperature, without stirring, until 2 minutespost-injection. The cuvette was then returned to the aggregometer well,a base-line being recorded for 2 minutes to detect any spontaneousaggregation. At exactly 4 minutes after injection of the evaluantsolution, 50 μl of the appropriate ADP or thrombin solution was injectedand aggregation recorded. Evaluants were studied, normally, at one ormore of the following final concentrations: 500 μM, 100 μM, 50 μM, and10 μM. Control experiments (e.g., ethanol in a final concentration of0.095% v/v) were performed in parallel with those involving therespective evaluants, and were initiated either 1 minute prior to or 1minute after the start of experiments employing the test compounds. Thispermitted injections to be made precisely at the specified times.Normally, 2 pairs of aggregations were carried out (at 70/71 and 80/81minutes post-venipuncture). Evaluant and control aggregations werestudied in alternate (Y₁ or Y₂) channels of the dual-channelaggregometer in order to detect any effects due to malfunction of aspecific channel.

The pH of a sample of the plasma employed in aggregometric studies wasroutinely measured at 37° C. after the injection of aggregation-stimulusat 70/71 minutes post-venipuncture. Also, the pH of a second plasmasample, maintained at 37° C. under 5% CO₂ -95% air mixture, wasdetermined at the conclusion of the 80/81 minute aggregation-pair (e.g.,90 minutes from venipuncture). The readings ranged from 7.5-7.7 in bothinstances. Plasma pH was not affected perceptibly by the addition ofrepresentative evaluants in concentrations employed in thisinvestigation.

In evaluating aggregometric tracings, primary attention was paid tointensity of aggregation, i.e., the maximum change in percentage oflight transmittance with special attention to any abolition ordimunition of the secondary and even the primary aggregation-waves.Platelet aggregation inhibitory potency was expressed as the % reductionin maximal aggregation intensity effected by the evaluant compound, withrespect to the maximal aggregation intensity evident in thecorresponding control experiment. In ADP-induced aggregationexperiments, as previously observed, the secondary aggregation wave wasnormally abolished by inhibitory responses of approximately 30% orhigher; effects on the primary aggregation wave were evident, normally,in inhibitions of 40% or stronger. (Cf. Roper et al, Am. J. Clin.Pathol., Vol. 71, pages 263-268 (1979)); Mills et al, Life Sci., Vol.14, pages 659-672 (1974); Newhouse and Clark, in Triplett (Ed.),Platelet Function: Laboratory Evaluation and Clinical Application,Chicago, American Society of Clinical Pathologists, 1978, pages109-121).

Relationships between the molecualr constitution of theevaluant-compounds and their inhibitory effects on platelet aggregation(Cf. Quintana et al, Thromb. Res., Vol. 22, pages 665-680 (1981);Quintana et al, Thromb. Res., Vol. 24, pages 379-395 (1981); Quintana etal, Chem-Biol. Interactions, Vol. 38, pages 135-144 (1982); Quintana etal, Biophys., J., Vol. 37, pages 130-133 (1982)) are summarized in Table2. Under conditions substantially comparable to those reported inThromb. Res., Vol. 22, pages 665-680 (1981), the compounds of theinvention are capable of inhibiting thrombin-induced aggregation withouteliminating thrombin-effected clotting.

                                      TABLE 2                                     __________________________________________________________________________    Relationships Between Chemical Constitution                                   and Inhibition of Human                                                       Blood Platelet Aggregation                                                                           % INHIBITION OF AGGREGATION BY                                                INDICATED CONCENTRATIONS OF                                                   EVALUANT COMPOUNDS                                                            ADP-INDUCED                                                                             THROMBIN-INDUCED                             EXAMPLE                                                                              COMPOUND        AGGREGATION                                                                             AGGREGATION                                  __________________________________________________________________________     2     α,α'-Bis(1,2,3,4-tetrahydroiso-                                                   0% at                                                                              50 μM                                                                           *                                                   quinolino)-p-xylene Dihydro-                                                  bromide                                                                 5     α,α'-Bis(6-hydroxy-1,2,3,4-                                                       21.5% at                                                                           50 μM                                                                           *                                                   tetrahydroquinolino)-p-xylene                                                 Dihydrobromide                                                          7     1,10-Bis(6-hydroxy-1,2,3,4-                                                                   25.9% at                                                                           50 μM                                                                           *                                                   tetrahydroquinolino)decane                                                    Dihydriodide                                                            9     1-Decyl-6-hydroxy-1,2,3,4-                                                                    33.0% at                                                                           50 μM                                                                           *                                                   tetrahydroquinolino)decane                                                    Hydriodide                                                             11     1-Hexyl-6-hydroxy-1,2,3,4-                                                                    45.9% at                                                                           100 μM                                                tetrahydroquinoline Hydrio-                                                                   37.2% at                                                                           50 μM                                                                           *                                                   dide                                                                   12     1-Tetradecyl-6-hydroxy-1,2,                                                                   0% at                                                                              50 μM                                                                           *                                                   3,4-tetrahydroquinoline                                                       Hydriodide                                                             13     N,N-Dibutylnicotinamide                                                                       0% at                                                                              100 μM                                                                          *                                                   (Intermediate)                                                         14     α,α'-Bis[3-(N,N-dibutyl-                                                          49.7% at                                                                           50 μM                                                                           *                                                   carbamoyl)piperidino]-p-                                                      xylene Dihydrobromide                                                  16     α,α'-Bis[3-(N,N-didecylcar-                                                       0% at                                                                              50 μM                                                                           *                                                   bamoyl)piperidino]-p-xylene                                                   Dihydrobromide                                                         19     α,α'-Bis[3-(N-decylcarba-                                                         0% at                                                                              50 μM                                                                           *                                                   moyl)piperidino]-p-xylene                                                     Dihydrobromide                                                         20     N-Methyl-N-octylnicotinamide                                                                  12.1% at                                                                           100 μM                                                                          *                                                   (Intermediate)                                                         21     N-Methyl-N-octylnipecotamide                                                                  49.9% at                                                                           100 μM                                                                          *                                            22     1-Methyl-3-(N-methyl-N-                                                                       33.1% at                                                                           100 μM                                                                          *                                                   octylcarbamoyl)piperidine                                                     Hydrochloride                                                          23     1-Methyl-3-(N,N-dibutylcar-                                                                   11.8% at                                                                           100 μM                                                                          *                                                   bamoyl)piperidine Hydriodide                                           25     1-Methyl-3-(N-decylcarbamoyl)-                                                                45.6% at                                                                           100 μM                                                                          *                                                   piperidine Hydriodide                                                  28     1,10-Bis[N-(1-methylnipecotoyl)-                                                              30.9% at                                                                           50 μM                                                                           *                                                   amino]decane Dihydriodide                                              31     1,6-Bis[N-(1-methylnipeco-                                                                    17.0% at                                                                           500 μM                                                                          *                                                   toyl)amino]hexane Dihydrio-                                                   dide                                                                   33     1,10-Bis(N-methylpiperidinium)-                                                               0% at                                                                              100 μM                                                                          *                                                   decane Diiodide                                                        35     N,N'-Bis(nipectotoyl)piperazine                                                               48.9% at                                                                           500 μM                                                                          *                                                   Dihydrobromide                                                         37     N,N'-Bis(1-decylnipecotoyl)-                                                                  93.8% at                                                                           50 μM                                                                           88.2% at 50 μM                                   piperazine Dihydriodide                                                                       48.3% at                                                                           10 μM                                          39     1,2-Bis(N-nipecotoyl-N-methyl-                                                                46.3% at                                                                           500 μM                                                                          *                                                   amino)ethane Dihydrobromide                                            40     1,2-Bis[N-(1-decylnipecotoyl)-                                                                82.6% at                                                                           50 μM                                                                           *                                                   N-methylamino]ethane                                                          Dihydriodide                                                           41     1,2-Bis[N-(1-hexylnipecotoyl)-                                                                71.6% at                                                                           50 μM                                                                           *                                                   N-methylamino]ethane                                                          Dihydriodide                                                           42     1,6-Bis[N-(1-decylnipecotoyl)-                                                                85.6% at                                                                           50 μM                                                                           88.3% at 50 μM                                   N-methylamino]hexane                                                          Dihydriodide                                                           43     1,6-Bis[N-(1-decylnipecotoyl)-                                                                38.6% at                                                                           50 μM                                                                           *                                                   N-butylamino]hexane                                                           Dihydriodide                                                           44     1,6-Bis[N-(1-hexylnipecotoyl)-                                                                72.4% at                                                                           50 μM                                                                           *                                                   N-butylamino] hexane                                                          Dihydriodide                                                           __________________________________________________________________________     *Not determined.                                                         

It would appear that the compounds of the invention are highly effectiveby penetrating the lipid bilayer of the platelet membrane and byinteracting as cations with negatively charged phospholipids (e.g.,phosphatidylserine and phosphatidylinositol) within the bilayer's innersegment. It would further appear that, in the event of such penetration,the cationic form of the compounds interferes with phospholipaseactivation by counteracting stimulus-induced mobilization of Ca++ ionsand Ca++-dependent phospholipase activity. It would also appear thatactivity is dependent upon (i) intramolecular distances between andcharge levels of pivotal atoms and/or functions, (ii) molecular geometryand flexibility, and (iii) hydrophobic characteristics of molecularsegments.

The data reflect that the compounds possess appropriate hydrophobiccharacter to penetrate the lipid bilayer of the platelet plasma membraneand, subsequently, are capable of generating sufficient quantities oftheir cationic species to counteract massively stimulus-inducedmobilization of Ca++ ions and, thereby restrain or void Ca++-dependentphospholipase activity. By means of this mechanism and their interactionwith negatively charged phospholipids (e.g., phosphoatidylserine andphosphatidylinositol) within the bilayer's inner segment, the compoundsfunction as effective membrane stabilizing agents. There iscorroborating evidence in the literature in support of thesecontentions. Vanderhoek and Feinstein (Vanderhoek et al, Mol.Pharmacol., vol. 16, pages 171-180 (1979)) present especially convincingdata and cite those of others [Sun et al, Lipids, Vol. 14, pages 229-235(1979): Rittenhouse-Simmons, J. Clin. Invest., Vol. 63, pages 580-587(1979)] in emphasizing the prominent function of Ca++ in controllingphospholipase-A₂ and phospholipase-C activity. The importance ofhydrophobic character, in imparting appropriate affinity for moleculesto consummate interaction leading to the prevention of Ca++mobilization, was stressed by Lullman et al [Lullman et al, Biochem.Pharmacol., Vol. 20, pages 2969-2974 (1980)].

The compounds described hereinabove useful for the inhibition of bloodplatelet aggregation could be administered orally, parenterally, orrectally.

Employing computed plasma concentrations for aspirin, dipyridamole andsulfinpyrazone, the most frequently used antithrombotic agents incontemporary practice, the most potent of the compounds of the inventionare effective at lower levels, with due consideration for variances (i)in the extent and rate of absorption, (ii) in biodistribution andprotein binding, and (iii) in the rate and diversity ofbiotransformation. Example 37, in Table 1, inhibits 93.8% ADP-inducedand 88.2% thrombin-elicited human blood platelet aggregation. Incomparison, at the same concentrations, aspirin effects 24% inhibitionin ADP- and 9% inhibition in thrombin-induced aggregation (see FIGS. 1and 2). The literature suggests an even lesser potency than aspirin fordipyridamole (10% at 250 μM concentration in ADP-induced aggregation;Cucuiani et al, J. Lab. Clin. Med., Vol. 77, pages 958-974 (1971) andsulfinpyrazone (0% at 2,470 μM concentrations in ADP-inducedaggregation; Packham et al, J. Exp. Med., Vol. 126, pages 171-188(1967)). Dipyridamole and sulfinpyrazone could exert their effectsaccording to different mechanisms, however. It should be added thatchlorpromazine, employed as one of the key reference compounds inexploratory work on platelets, is much less effective inthrombin-induced aggregation that the piperidine derivatives discussedabove. It is, also, considerably less effective in ADP-inducedaggregation. (See FIG. 2).

The structure of the compounds of this invention would suggest a lowertoxicity that those currently in use. Even aspirin's adverse effects aresevere enough to counsel against its use by survivors of myocardialinfarction (NHLBI-AMISRF, J. Am. Med. Assoc., Vol. 243, pages 661-669(1980). Indeed, the compounds of this invention would appear to yieldless toxic metabolites in the process of their biotransformation. Inthat respect, piperidine is known to be a natural metabolite andcomparatively high quantities have been reported to occur in man(excretion in urine about 3 to 20 mg/day) (Williams, DetoxicationMechanisms, 2nd Edition, New York, John Wiley and Sons, 1959, page 567).Piperidine-3-carboxylic acid (nipecotic acid) has not been discerned tohave deleterious effects (Johnston, Ann. Rev. Pharmacol. Toxicol., Vol.18, pages 269-289 (1978); cf. Krogsgaard-Larsen and Johnston, J.Neurochem., Vol. 25, pages 797-802 (1975)). Nicotinic acid (niacin), itsaromatic analog, along with nicotinic acid amide (niacinamide), areknown metabolites. Aminoalkanes or aralkanes which could be generatedfrom some compounds of the invention are generally not known to beconverted into harmful products (Williams, Detoxication Mechanisms, 2ndEdition, New York, John Wiley and Sons, 1959, pages 128-139 and 146-147).

In broader terms of preventive medicine, it may be preferable to inhibitthe adhesion-release-aggregation chain reaction at its very inceptionwith platelet membrane stabilizing agents by precluding activities ofphospholipases. This is especially true if it is considered thatplatelet cyclo-oxygenase inhibitors like aspirin incapacitate thepatient's platelets for the rest of their life span (Walder et al, Mol.Pharmacol., Vol. 13, pages 407-414 (1977)) and could possibly result inthe concurrent inhibition of endothelial cyclo-oxygenase which wouldreduce or block generation of the endogenous aggregation inhibitorprostacyclin (PGI₂) (Moncada et al, in Gilman et al (Eds.), ThePharmacological Basis of Therapeutics, 6th Edition, New York, MacMillan,1980, page 669; cf. Harris et al, Ann. Rev. Physiol., Vol. 41, pages653-668 (1979)).

Within the context of these considerations, the compounds of theinvention could be formulated with suitable pharmaceutically acceptablecarriers into a unit dosage form containing from about 28 to about 286milligrams of active ingredient. Accordingly, orally administereddosages in the range of from about 0.38 to about 3.8 mg/kg of bodyweight per adult animal, every six hours, would be sufficient to inhibitblood platelet aggregation. For infant or young animals, dosages in therange of from about 0.08 to about 0.76 mg/kg would be sufficient.Obviously, parenteral administration should reduce the referencedquantities, and rectal administration could also require a modificationin the dosage.

We claim:
 1. The compound N,N'-bis(nipecotoyl)piperazine dihydrobromide.2. The compound N,N'-bis(1-decylnipecotoyl)piperazine dihydriodide.
 3. Apharmaceutical composition in unit dosage form suitable foradministration to an animal in need thereof comprising apharmaceutically acceptable carrier and a blood platelet aggregationinhibiting amount of a compound as in any one of claims 1 or
 2. 4. Amethod for the inhibition of blood platelet aggregation comprisingadministering to an animal in need thereof a blood platelet aggregationinhibiting amount of a compound as in any one of claims 1 or 2.